Recombinant Enterococcus faecalis Elongation factor Tu (tuf)

What is Elongation Factor Tu in Enterococcus faecalis?

Elongation factor Tu (EF-Tu) in E. faecalis is a GTP binding protein that plays a central role in protein synthesis. It functions as an essential and universally conserved GTPase that ensures translational accuracy by mediating the recognition and transport of aminoacyl-tRNAs and their positioning to the A site of the ribosome. This protein is encoded by tuf genes, with E. faecalis specifically carrying a single tufA gene, unlike some other enterococcal species that possess two tuf gene variants (tufA and tufB) .

How does E. faecalis EF-Tu differ from EF-Tu in other enterococcal species?

E. faecalis possesses only the tufA gene variant, while 11 other enterococcal species (including E. avium, E. casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, and E. raffinosus) harbor both tufA and tufB genes. Phylogenetic analysis demonstrates that E. faecalis and other single-tuf enterococcal species diverged from the enterococcal lineage before the common ancestor of the dual-tuf species. This genetic difference suggests that the acquisition of the second tuf gene (tufB) occurred through horizontal gene transfer from an ancestral streptococcus or streptococcus-related species to the common ancestor of the 11 dual-tuf enterococcal species .

What are the structural components of E. faecalis EF-Tu?

E. faecalis EF-Tu, like other bacterial EF-Tu proteins, consists of three functional domains. Domain I (approximately amino acids 1-200) forms a helix structure with Rossmann fold topology, which is a structural motif found in proteins that bind nucleotides. This domain houses the GTP/GDP binding regions essential for its function in protein synthesis. Domains II (approximately amino acids 209-299) and III (approximately amino acids 301-393) are largely composed of beta sheets. Together, these domains create the functional architecture that enables EF-Tu to perform its canonical and moonlighting functions .

How can researchers amplify and sequence the tuf gene from E. faecalis?

Researchers can amplify the tuf gene from E. faecalis using degenerate PCR primers derived from consensus sequences. For example, the primer pair U1 (5′-AAYATGATIACIGGIGCIGCICARATGGA-3′) and U3 (5′-CCIACIGTICKICCRCCYTCRCG-3′) can be used to amplify an 886-bp portion of the tuf gene. For E. faecalis specifically, direct sequencing of PCR products has proven effective. Alternatively, researchers can use species-specific primers such as EntA1 (5′-ATCTTAGTAGTTTCTGCTGCTGA-3′) and EntA2 (5′-GTAGAATTCAGGACGGTAGTTAG-3′) to amplify E. faecalis tuf gene fragments with greater specificity .

What evidence supports horizontal gene transfer in the evolution of enterococcal tuf genes?

Multiple lines of evidence support horizontal gene transfer in the evolution of enterococcal tuf genes:

• Phylogenetic analysis shows that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene (found in 11 species but not E. faecalis) clusters with the genera Streptococcus and Lactococcus.

• Primary structure analysis revealed four amino acid residues within the sequenced regions that are conserved and unique to both the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis.

• The 11 enterococcal species possessing two tuf genes share a common ancestor, while E. faecalis and five other species with only one tufA copy diverged earlier in the enterococcal lineage.

These findings strongly suggest that an ancestral streptococcus or streptococcus-related species horizontally transferred a tuf gene to the common ancestor of the dual-tuf enterococcal species .

How can researchers confirm the presence and copy number of tuf genes in enterococcal genomes?

Researchers can confirm the presence and copy number of tuf genes in enterococcal genomes through Southern hybridization. This involves:

• Digesting genomic DNA samples (1-2 μg) to completion with appropriate restriction endonucleases (e.g., BglII and XbaI).

• Preparing DIG-labeled probes using recombinant plasmids carrying either tufA or tufB sequences.

• Performing hybridization according to standard protocols.

This approach can definitively determine whether a species contains one or two copies of the tuf gene, confirming the results obtained through PCR and sequencing analyses .

What expression systems are optimal for producing recombinant E. faecalis EF-Tu?

For optimal expression of recombinant E. faecalis EF-Tu, an E. coli expression system using the pET vector series is recommended. The protocol should include:

• Amplification of the complete E. faecalis tufA gene using high-fidelity polymerase and primers containing appropriate restriction sites.

• Cloning into a pET vector with a His-tag for purification.

• Transformation into an expression strain such as BL21(DE3).

• Induction with IPTG (0.5-1 mM) when cultures reach OD600 of 0.6-0.8.

• Expression at lower temperatures (16-25°C) overnight to enhance protein folding.

This approach typically yields functional recombinant EF-Tu with its GTPase activity preserved, which is crucial for functional studies .

What purification strategies yield the highest purity recombinant E. faecalis EF-Tu?

A multi-step purification strategy is recommended for obtaining high-purity recombinant E. faecalis EF-Tu:

• Initial capture using Nickel-NTA affinity chromatography:

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

  • Elute with an imidazole gradient (50-300 mM)

• Intermediate purification using ion-exchange chromatography:

  • Dialyze affinity-purified protein against buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl

  • Apply to a Q-Sepharose column and elute with a NaCl gradient (50-500 mM)

• Polishing step using size-exclusion chromatography:

  • Apply concentrated protein to a Superdex 75 or 200 column equilibrated with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl

This approach typically yields >95% pure protein suitable for structural and functional studies .

How can researchers assess the functional activity of purified recombinant E. faecalis EF-Tu?

The functional activity of purified recombinant E. faecalis EF-Tu can be assessed through multiple complementary approaches:

• GTPase activity assay:

  • Measure GTP hydrolysis rates using a malachite green phosphate detection system or radiometric assays with [γ-32P]GTP

  • Reaction conditions: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM KCl, 0.5 mM GTP, and 1-5 μM purified EF-Tu

• Aminoacyl-tRNA binding assay:

  • Evaluate binding to aminoacyl-tRNA using filter binding assays with radiolabeled tRNAs

  • Alternatively, use fluorescence anisotropy with fluorescently labeled tRNAs

• Ribosome interaction studies:

  • Assess interaction with ribosomes using sucrose gradient centrifugation or surface plasmon resonance

These assays provide comprehensive evaluation of the canonical functions of EF-Tu in protein synthesis .

How does E. faecalis EF-Tu function as a moonlighting protein?

E. faecalis EF-Tu functions as a moonlighting protein by performing additional roles beyond its canonical function in protein synthesis. Based on studies of EF-Tu in related bacteria, it can:

• Localize to the bacterial cell surface despite lacking classical secretion signals, where it can interact with host molecules.

• Bind to various host proteins, including plasminogen, fibronectin, and complement factors.

• When bound to plasminogen, facilitate its conversion to plasmin in the presence of plasminogen activators, potentially contributing to bacterial invasion.

• Undergo processing events on the cell surface that may generate fragments retaining specific binding capabilities to host proteins.

These moonlighting functions may contribute to the virulence and pathogenicity of E. faecalis, particularly in its interactions with host tissues during infection .

What structural features of E. faecalis EF-Tu facilitate its moonlighting activities?

Several structural features of E. faecalis EF-Tu likely facilitate its moonlighting activities:

• The accumulation of positively charged amino acids in short linear motifs (SLiMs), which can mediate interactions with host molecules.

• Post-translational modifications and protein processing events that expose binding sites normally buried in the protein's interior.

• Domain organization that allows different regions of the protein to engage in distinct interactions:

  • Domain I (with its Rossmann fold) may interact with nucleotides and nucleic acids

  • Domains II and III likely mediate protein-protein interactions with host molecules

Bioinformatics and structural modeling studies indicate that these features promote multifunctional behavior. Additionally, the A+T rich genome characteristic of many bacteria influences codon bias, which may contribute to the accumulation of positively-charged residues in SLiMs that mediate host interactions .

How can researchers investigate the interaction between recombinant E. faecalis EF-Tu and host proteins?

Researchers can investigate interactions between recombinant E. faecalis EF-Tu and host proteins using several complementary techniques:

• Solid-phase binding assays:

  • Immobilize purified host proteins (e.g., plasminogen, fibronectin) on microtiter plates

  • Add varying concentrations of recombinant EF-Tu

  • Detect binding using EF-Tu-specific antibodies in an ELISA-like format

• Surface plasmon resonance (SPR):

  • Immobilize either EF-Tu or the host protein on a sensor chip

  • Measure real-time binding kinetics and determine association/dissociation constants

  • Evaluate the effects of pH, ionic strength, and temperature on binding interactions

• Pull-down assays:

  • Use His-tagged EF-Tu with Ni-NTA resin to capture interacting host proteins

  • Alternatively, use biotinylated host proteins with streptavidin beads

  • Identify bound proteins by western blotting or mass spectrometry

• Functional assays for specific interactions:

  • For plasminogen interactions: Measure plasmin generation using chromogenic substrates

  • For adhesion-related functions: Assess binding to host cell lines using fluorescently labeled EF-Tu

These methods provide both qualitative and quantitative data on EF-Tu's interactions with host molecules .

How can recombinant E. faecalis EF-Tu be used to study pathogenicity mechanisms?

Recombinant E. faecalis EF-Tu can serve as a valuable tool for studying pathogenicity mechanisms through several research approaches:

• Construction of domain-specific mutants:

  • Generate recombinant EF-Tu variants with mutations in specific domains

  • Evaluate how these mutations affect binding to host molecules and pathogenicity

  • Identify critical residues involved in moonlighting functions

• Inhibition studies:

  • Use recombinant EF-Tu or specific fragments as competitors in infection models

  • Determine if pre-blocking host receptors with recombinant EF-Tu reduces bacterial adherence or invasion

  • Develop potential therapeutic approaches targeting EF-Tu-host interactions

• Immunological studies:

  • Assess the immunogenicity of recombinant EF-Tu in animal models

  • Evaluate if antibodies against EF-Tu provide protection against E. faecalis infection

  • Study the inflammatory responses triggered by EF-Tu interaction with host immunity

• Cross-species comparison:

  • Compare the properties of recombinant EF-Tu from E. faecalis with those from other enterococcal species

  • Investigate whether differences in EF-Tu structure correlate with varying pathogenicity among species

These approaches can reveal how EF-Tu contributes to bacterial virulence beyond its canonical role in protein synthesis .

What role might E. faecalis EF-Tu play in horizontal gene transfer mechanisms?

While the tuf gene itself has been subject to horizontal gene transfer in enterococcal evolution, the EF-Tu protein might also facilitate horizontal gene transfer through several potential mechanisms:

• DNA binding capability:

  • EF-Tu's nucleotide-binding domain might enable interaction with extracellular DNA

  • This could promote DNA uptake during natural transformation

• Association with mobile genetic elements:

  • EF-Tu may interact with proteins encoded by pathogenicity islands or conjugative plasmids

  • For example, E. faecalis harbors a pathogenicity island (PAI) of 153 kb containing virulence factors

• Surface localization:

  • As a surface-exposed protein, EF-Tu could facilitate cell-to-cell contact

  • This might enhance conjugative transfer of genetic elements

• Interaction with transfer machinery:

  • EF-Tu might interact with components of conjugation systems

  • Potentially, it could modulate transfer frequencies of mobile genetic elements

Research to test these hypotheses would involve creating EF-Tu knockout strains and assessing their gene transfer capabilities compared to wild-type strains .

How do post-translational modifications affect E. faecalis EF-Tu function?

Post-translational modifications likely play significant roles in regulating E. faecalis EF-Tu functions, particularly its moonlighting activities:

• Proteolytic processing:

  • EF-Tu can undergo multiple processing events on the bacterial cell surface

  • N-terminomics pipelines have characterized these processing events in related bacteria

  • Fragments of EF-Tu retain binding capabilities to host proteins

  • Processing may expose cryptic binding sites normally buried within the protein structure

• Phosphorylation:

  • Phosphorylation of specific threonine and serine residues may regulate GTPase activity

  • Modifications may also affect surface exposure and interactions with host molecules

  • Research methods include phosphoproteomic analysis using mass spectrometry

• Methylation and acetylation:

  • These modifications may alter protein-protein interactions

  • They could influence the protein's subcellular localization and extracellular functions

  • Techniques to study these include mass spectrometry and specific antibodies against modified forms

• Glycosylation:

  • Surface proteins in gram-positive bacteria can be glycosylated

  • Glycosylation may protect against proteolytic degradation and modulate host interactions

  • Detection methods include periodic acid-Schiff staining and lectin binding assays

Understanding these modifications is critical for comprehending the full functional repertoire of EF-Tu in bacterial physiology and host-pathogen interactions .

What are common challenges in expressing recombinant E. faecalis EF-Tu and how can they be addressed?

These solutions address the most frequent challenges encountered during recombinant EF-Tu production while preserving the protein's functional integrity for downstream applications .

How can researchers differentiate between canonical and moonlighting functions in experimental designs?

Differentiating between canonical and moonlighting functions of E. faecalis EF-Tu requires careful experimental design:

• Domain-specific mutations:

  • Generate variants with mutations in the GTP-binding pocket that disrupt canonical function

  • Test if these variants retain moonlighting activities like host protein binding

  • Similarly, create mutations in surface-exposed regions predicted to mediate host interactions

  • Determine if these affect moonlighting functions while preserving canonical activity

• Subcellular localization studies:

  • Use fractionation techniques to separate cytoplasmic and surface-associated EF-Tu

  • Analyze each fraction for canonical and moonlighting activities

  • Employ immunofluorescence microscopy with anti-EF-Tu antibodies to visualize localization

• Competitive inhibition experiments:

  • Use domain-specific antibodies or peptides to block different regions of EF-Tu

  • Assess which functions are inhibited by each blocking agent

  • This approach can map functional domains to specific activities

• Time-course studies:

  • Monitor translational activity versus surface exposure during different growth phases

  • Determine if moonlighting functions predominate under specific conditions

  • This can reveal regulatory mechanisms controlling functional switching

These approaches allow researchers to dissect the multifunctional nature of EF-Tu and understand how one protein performs distinct roles in different cellular contexts .

What controls should be included when studying E. faecalis EF-Tu interactions with host proteins?

When studying E. faecalis EF-Tu interactions with host proteins, several essential controls should be included:

• Negative controls:

  • Unrelated bacterial proteins of similar size/structure to rule out non-specific binding

  • Heat-denatured EF-Tu to determine if native conformation is required

  • Binding assays in the presence of high salt concentrations to test electrostatic contributions

• Specificity controls:

  • Competition assays with unlabeled EF-Tu to demonstrate saturability

  • Pre-blocking with antibodies against specific EF-Tu domains

  • Testing host proteins from different species to assess evolutionary conservation

• Domain-specific controls:

  • Individual recombinant domains of EF-Tu to map interaction sites

  • Chimeric proteins with domains from non-binding EF-Tu homologs

  • Site-directed mutants targeting potential interaction residues

• Functional validation:

  • For plasminogen interactions: include plasminogen activator inhibitors

  • For adhesion functions: include enzymatic treatments of host cells to remove specific receptors

  • In infection models: compare wild-type strains with tuf mutants or strains expressing modified EF-Tu

This comprehensive control strategy ensures that observed interactions are specific, biologically relevant, and correctly attributed to particular structural features of EF-Tu .